Carnitine palmitoyltransferase (CPT) activity is dually located on both the outer and inner surfaces of the inner mitochondrial membrane. The activity located on the outer surface has been proposed as the rate-limiting enzyme in the hepatic mitochondrial oxidation of long-chain fatty acids and, thus, is pivotal in regulation of hepatic energy metabolism, particularly in states such as diabetes, starvation, neonatal transition or where diets high in fat are consumed. Little is known about the properties of the inner enzyme due to the difficulties of studying either enzyme independently once the mitochondria are lysed. The use of intact mitochondria and inverted submitochondrial vesicles has allowed the study of each enzyme, in situ, independently. However, these methods do not delineate whether the inner and outer activities arise from the same enzyme protein, what the specific membrane lipid requirements are for activity, how the enzymes are oriented within the membrane, or which enzyme is induced in diabetes, starvation, neonatal transition or with high fat diets, or how regulation is, indeed, accomplished. Thus, our specific aims are: 1. Determine whether CPT-A and CPT-B are the same protein. Determine the amino acid sequence of the outer enzyme (CPT-A) and the inner enzyme (CPT-B) and predict the orientation in the membrane. 2. Detemine the lipid dependencies and orientation of CPT-A and CPT-B using defined lipid mixtures in a reconstitued system. Carnitine palmitoyltransferase is a focal regulatory enzyme in the control of hepatic metochondrial long chain fatty acid oxidation. In spite of its central role in physiologic and pathophysiologic conditions, relatively little is known about the regulation of this enzyme. In addition, genetic CPT deficiency has been described, but the underlying basis of deficiency is unknown. These studies will aid in the delineation of regulation of this enzyme. Our ultimate goal is to determine the regulation of CPT at the genomic level. A fundamental question in biochemistry will be addressed when we ascertain how protein(s) catalyzing the same reaction are inserted in controlled fashion on two sides of the same membrane.